A436 Biochemical Society Transactions (2000) Volume 28, Part 5
1592Molecular Modeling Design Strategy o n Novel Drugs for HIV-1 Tat/TAR Complex L.J.O. Figueiredo, O.A.C. Antunes
Dep. Bioquimica, Instituto de Quimica, UFRJ, DBBM/IOC/FIOCRUZ, Lab. Bioquimica de Proteinas e Peptideos, Dep.Quimica Inorganica, IQ/UFRJ, Far- ManguinhosFIOCRUZ
1594 MODULATION O F NF-kB ACTIVATION BY TARGETED DELIVERY O F DEXAMETHASONE TO MACROPHAGES M,MAG"I.R. CRINELLI, A. ANTONELLI, M. BIANCHI, L. GENTILINI, S. SCARAMUCCI
ISTITUTO DI CHIMICA BIOLOGICA iG.FORNAINIi, VIA SAFFI N. 2,61029 URBINO, ITALE E-MAIL:
[email protected] Glucocorticoids are a widely used class of anti-inflammatory and immunosuppressive drugs, but their therapeutic use is limited by endocrine and metabolic side effects that they produce when given systemically. Since cells of monocyte/macrophage lineage play an important role in the pathogenesis of several autoimmune and inflammatory diseases, a drug-delivery system which targets phagocytic cells was studied. We had previously demonstrated that Dexamethasone, a potent glucocorticoid analogue, can be encapsulated in erythrocytes and selectively delivered to macrophages (Magnani et al., Drug Delivery, 2151-155, 1995). In addition, lipopolysaccharide (LPS) stimulation of Dexamethasone-targeted macrophages results in the suppression of TNF-a secretion (D'Ascenzo et al., Erythrocytes us Drug Carrier in Medicine, 81-88, 1997). Here we demonstrate that Dexamethasone administration to macrophages by means of opsonized RBC allow to efficiently interfere with NF-kB activation. This NF-kB repression was in part mediated by induction of IkBa gene transcription and, as a consequence, by an increased rate of IkBa protein synthesis. Addition of LPS to macrophages targeted with Dexamethasoneloaded RBC resulted in a significant residual amount of IkBa and in a 40% inhibition of the NF-kB DNA binding activity. Furthermore, NF-kB inactivation correlated with down-modulation of TNF-a mRNA expression, demonstrating that suppression of TNF-a production in Dexamethasone targeted cells occurs at the transcriptional level. Although the same molecular mechanisms are involved in the immunosuppressive activity of free or RBC-loaded Dexamethasone, this second system of drug delivery offers the advantage of being extremely selective for cells of the monocyte/macrophage lineage.
595 High Affinity, Slow Binding FXa Inhibitors Are Poor 593 Mutations in Human P O N l : Role in Organophosphorus Sensitivity and Vascular Disease
-1,
V H Brophy2, LG Costal, RJ Richte?, T Hagen*,
D M Shih3, AJ Lusis3, G P Jarvik*, C E Furlong2
Departments of Environmental Health, and 2Medicine, University of Washington, Seattle, WA 98195; 3Department of Medicine, UCLA School of Medicine, Los Angeles, C A 90091 Two common polymorphisms have been found in the H D L associated enzyme P O N l , L55M and Q192R. In addition, we recently identified 5 mutations in the 5' non-coding region of the PONl gene. Studies on the functional consequences of these mutations have involved experiments with PONI-knockout mice (missing both liver and plasma PON1) and determination of P O N l status (genotype and phenotype) in patients with carotid artery disease (CAAD). Knockout mice are highly sensitive to the oxon forms of diazinon and chlorpyrifos, but not parathion. They are also susceptible to vascular disease if fed a high fat diet. Reconstitution of plasma P O N l with each of the purified human PONl192 isoforms showed that plasma P O N l alone provides protection against diazoxon and chlorpyrifos oxon but not paraoxon. Levels of plasma P O N l are important in determining resistance to OP compounds. Determination of P O N l status in patients with CAAD showed lower levels of P O N l in patients compared with controls. Analysis of the polymorphisms 5' to the PONl coding region indicated that a G to A substitution at position -162 affects the level of expressed P O N l . Reporter gene constructs show a two-fold difference in expression with this single base change. These results support the concept that it is as important to determine levels of P O N l as it is to determine sequence differences between individuals. N I H Grants ES09883, ES07033, ES09601/EPR-R826886, HL3300568.
0 2000 Biochemical Society
Inhibitors Of Clot Formation in vitro H Martin, J Mahler, S Dyas, T Auton & J Liebeschuetz Protherics Molecular Design Limited, Beechfield House, Lyme Green Business Park, Macclesfield, Cheshire. SK11 OJL Factor Xa is a trypsin-like serine protease and plays a central role in blood coagulation by cleaving thrombin which converts fibrinogen to fibrin. Inhibitors of FXa are being developed as an approach to the clinical regulation of haemostasis. Some small molecule, reversible, competitive inhibitors of thrombin (1) and FXa (2) have been shown to be slow binders. In these examples, a 2-step inhibition mechanism was proposed a weak, rapid initial binding followed by a slow rearrangement of the protease leading to a tight enzyme/inhibitor complex. Slow binding compounds may be unattractive as anti-coagulant drugs since they give rise to steep doseresponse curves in anti-thrombotic models (3). We have used Computer-Aided Molecular Design t o help identify several series of compounds which are potent, non-covalent inhibitors of FXa. Certain members of one series had high affinity yet demonstrated slow binding characteristics and also had low activity in the prothrombin assay. e.g. PMD3330 was a potent FXa inhibitor (K; 4nM) with a slow on-rate (Kon 0.057nM-l.min-l). In the prothrombin time (PT) assay PMD3330 was a poor inhibitor of clot formation In another of our series of FXa inhibitors, slow
(PTIC50 66pM).
binding was not observed E.g. PMD3131 (K; 7.5nM, kon 0.43nMl m i n - l , PT IC50 1.IpM. These data suggest that both high affinity and fast binding may be necessary features of anti-thrombotic FXa inhibitors. 1) T Nilsson et al. J. Enz. Inhibit. 1998, 11-29. 2) A Betz et al. Biochemistry 1999,& 14582-14591. 3) M Elg et al. Thromb. Res. 1999, $?-4, 187-197.
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